Opiate receptors recognize endogenous opioid peptide neurotransmitters and exogenous opiate drugs of high abuse liability and analgesic benefits. Opiate receptors produce their effects in cells by altering intracellular pools of G-protein linked second messengers and activities of G-protein-linked ion channels. In order to improve understanding these key elements in mediating opiate addictions, these workers have used cloned opiate receptor cDNAs and in vitro mutagenesis to identify specific portions of the morphine-preferring m opiate receptor important for its expression, recognition of agonist and antagonist ligands from peptide and nonpeptide classes, and coupling to second messenger systems. During this year, studies of the mu receptor mutants that have served to help define amino acids important for recognition of peptide and nonpeptide agonists and antagonists have continued. Substantial mutagenesis work has extended work on the importance of a histidine residue in transmembrane domain VI of the mOR for the ligand binding activity. Substitution of His-297 with asparagine or glutamine retained high affinity binding of several opiate alkaloid and opioid peptide agonists and antagonists, while affinity for such ligands was drastically curtailed by replacement with alanine, leucine, aspartic acid, glutamic acid, arginine, lysine or phenylalanine. These and results of alkylation studies suggest critical roles for the length and hydrogen-bonding ability of the His-297 sidechain, and possibly, its orientation toward a hydrophilic receptor ligand-binding cavity. Deletion mutagenesis of rat and human 3'-untranslated regions (UTR) yielded approximately 5-fold increases in binding of m-selective radioligands. Serial deletion mapping of this region suggests that no specific sequence is required to negatively influence receptor expression, and that regions immediately upstream of the stop codon also affect expression. Current efforts are directed toward identifying the mechanism of this effect (i.e., transcriptional or translational). In order to evaluate the putative role of the 3' untranslated region (3' UTR) of the m opiate receptor in the regulation of its expression, several deletion constructs have been generated and tested in for [3H]DAMGO binding. These studies have revealed that deletions to the 3'-UTR of the m opiate receptor cDNA dramatically increases Bmax values for [3H]DAMGO binding. One construct, which deletes all but 30 nucleotides of 3'-UTR from the cDNA, shows up to 6-fold increases in receptor number. Co-transfection of the COS cells with another reporter gene, B-galactosidase, indicates that these dramatic alterations in m opiate receptor expression are not transfection dependent. Northern analysis of m opiate receptor mRNA expression in the transfected COS cells shows no differences in the level of message expressed when constructs with a full-length 3'-UTR are compared to the truncated constructs. These results suggest that stability of the mRNA could be contributing to the changes in receptor expression or that some secondary structure in the mRNA is being altered in the 3'-UTR truncated constructs.